SH-It happens: S-H bonds as intrinsic 2D-IR labels in proteins.

Cysteine S-H bonds have a spectroscopically convenient stretching frequency of ∼2550 cm-1. However, their cross section is low, and the band can be strongly broadened in heterogeneous environments, making detection very challenging. With two-dimensional infrared (2D-IR) setups achieving ever higher sensitivities in recent years, systematic use of the weak cysteine sulfhydryls (Cys-SHs) absorption band is now within reach, even at low millimolar protein concentrations. Here, we demonstrate the capabilities of Cys-SH as an intrinsic 2D-IR label in pyruvate oxidase from E. coli, an enzyme with ten cysteines in its native sequence. 1D-IR measurements on the wild-type and individual cysteine knock-out variants show that two such residues have especially narrow SH signatures, caused by their intrahelical hydrogen bonding. 2D-IR analysis of these bands reveals an extraordinarily high anharmonicity (∼110 cm-1) and a long vibrational lifetime (∼4 ps). This allows monitoring spectral diffusion via center line slope analysis for up to 10 ps-separately for both the ground and excited states. The unique spectroscopic features and its ease of introduction make Cys-SH a useful IR spectroscopic label.

Physician cooperation in outpatient cancer care. An amplified secondary analysis of qualitative interview data.

The importance of outpatient cancer care services is increasing due to the growing number of patients having or having had cancer. However, little is known about cooperation among physicians in outpatient settings. To understand what inter- and multidisciplinary care means in community settings, we conducted an amplified secondary analysis that combined qualitative interview data with 42 general practitioners (GPs), 21 oncologists and 21 urologists that mainly worked in medical practices in Germany. We compared their perspectives on cooperation relationships in cancer care. Our results indicate that all participants regarded cooperation as a prerequisite for good cancer care. Oncologists and urologists mainly reported cooperating for tumour-specific treatment tasks, while GPs' reasoning for cooperation was more patient-centred. While oncologists and urologists reported experiencing reciprocal communication with other physicians, GPs had to gather the information they needed. GPs seldom reported engaging in formal cooperation structures, while for specialists, participation in formal spaces of cooperation, such as tumour boards, facilitated a more frequent and informal discussion of patients, for instance on the phone. Further research should focus on ways to foster GPs' integration in cancer care and evaluate if this can be reached by incorporating GPs in formal cooperation structures such as tumour boards.

Evaluation of coverall field dry aerosol decontamination methods using a manikin.

A full-size manikin dressed in fire-resistant coveralls coated in 120 g of sodium bicarbonate was randomly given one of three treatments for dry aerosol decontamination. The three treatments were high-efficiency particulate air (HEPA) vacuum, a commercially available air shower, and the no treatment control. Immediately after the treatment, the coveralls were doffed and an air sample was taken in the breathing zone of the manikin to estimate airborne total and respirable dust concentrations to an unprotected worker post decontamination. Each treatment was applied four times for a total of 12 trials. Using analysis of variance (ANOVA) with alpha =.05 and Tukey's Honestly Significant Difference multiple comparison post-test, it was determined that HEPA vacuuming was not significantly different from the air shower for respirable dust, but only the air shower was significantly better than no decontamination (p =.037). For total dust, HEPA was not significantly different from the air shower, but both were significantly better than no treatment (p =.007, p =.004, respectively).

Species distribution models contribute to determine the effect of climate and interspecific interactions in moving hybrid zones.

Climate is a major factor delimiting species' distributions. However, biotic interactions may also be prominent in shaping geographical ranges, especially for parapatric species forming hybrid zones. Determining the relative effect of each factor and their interaction of the contact zone location has been difficult due to the lack of broad scale environmental data. Recent developments in species distribution modelling (SDM) now allow disentangling the relative contributions of climate and species' interactions in hybrid zones and their responses to future climate change. We investigated the moving hybrid zone between the breeding ranges of two parapatric passerines in Europe. We conducted SDMs representing the climatic conditions during the breeding season. Our results show a large mismatch between the realized and potential distributions of the two species, suggesting that interspecific interactions, not climate, account for the present location of the contact zone. The SDM scenarios show that the southerly distributed species, Hippolais polyglotta, might lose large parts of its southern distribution under climate change, but a similar gain of novel habitat along the hybrid zone seems unlikely, because interactions with the other species (H. icterina) constrain its range expansion. Thus, whenever biotic interactions limit range expansion, species may become 'trapped' if range loss due to climate change is faster than the movement of the contact zone. An increasing number of moving hybrid zones are being reported, but the proximate causes of movement often remain unclear. In a global context of climate change, we call for more interest in their interactions with climate change.

Kneelike structure in the spectrum of the heavy component of cosmic rays observed with KASCADE-Grande.

We report the observation of a steepening in the cosmic ray energy spectrum of heavy primary particles at about 8×10(16) eV. This structure is also seen in the all-particle energy spectrum, but is less significant. Whereas the "knee" of the cosmic ray spectrum at 3-5×10(15) eV was assigned to light primary masses by the KASCADE experiment, the new structure found by the KASCADE-Grande experiment is caused by heavy primaries. The result is obtained by independent measurements of the charged particle and muon components of the secondary particles of extensive air showers in the primary energy range of 10(16) to 10(18) eV. The data are analyzed on a single-event basis taking into account also the correlation of the two observables.

Choice of living arrangements.

BACKGROUND: The rights to choose where and with whom to live are widely endorsed but commonly denied to adults with intellectual disabilities (ID). The current study provides a contemporary benchmark on the degree of choice exercised by adult service users in the USA. METHOD: Data came from the National Core Indicators programme. Participants were 6778 adult service users living in non-family-home service settings in 26 US states. RESULTS: Most adults with ID did not participate in choosing where and with whom to live. Those with more support needs because of more severe ID and/or co-occurring conditions experienced less choice regarding living arrangements. Individuals living in their own home or an agency-operated apartment were more likely to choose where and with whom to live than individuals in nursing homes, institutions or group homes. However, few individuals with severe or profound ID chose where and with whom to live regardless of where they lived. CONCLUSIONS: In 2008, despite community-living policies that emphasise choice, many adult service users with ID in the USA experienced little or no choice about where and with whom to live, especially those individuals with more severe ID. Our findings provide a clear endorsement of policies promoting more individualised living settings, such as one's own home or an agency apartment, because these settings do provide substantially more choice about living arrangements.

Induction Patterns of an Extensin Gene in Tobacco upon Nematode Infection.

When sedentary endoparasitic nematodes infect plants, they induce complex feeding sites within the root tissues of their host. To characterize cell wall changes induced within these structures at a molecular level, we studied the expression of an extensin gene (coding for a major structural cell wall protein) in nematode-infected tobacco roots. Extensin gene expression was observed to be induced very early upon infection. This induction was weak, transient, and probably due to wounding during penetration and migration of the tobacco cyst nematode Globodera tabacum ssp solanacea-rum. In contrast, high extensin gene expression was observed during the whole second larval stage (an ~2-week-long phase of establishment of the feeding site) of the root knot nematode Meloidogyne javanica. During later stages of this interaction, expression gradually decreased. Extensin gene expression was found in at least three different tissues of the gall. We propose that distinct mechanisms lead to induced expression in these different cell types. The significance of these results for the understanding of plant-nematode interactions as well as the function of structural cell wall proteins, such as extensin, is discussed.

Increase of GKLF messenger RNA and protein expression during progression of breast cancer.

Genetic alterations found in carcinomas can alter specific regulatory pathways and provide a selective growth advantage by activation of transforming oncogenes. A subset of these genes, including wild-type alleles of GLI or c-MYC, and activated alleles of RAS or beta-catenin, exhibit transforming activity when expressed in diploid epithelial RK3E cells in vitro. By in vitro transformation of these cells, the zinc finger protein GKLF/KLF-4 was recently identified as a novel oncogene. Although GKLF is normally expressed in superficial, differentiating epithelial cells of the skin, oral mucosa, and gut, expression is consistently up-regulated in dysplastic epithelium and in squamous cell carcinoma of the oral cavity. In the current study, we used in situ hybridization, Northern blot analysis, and immunohistochemistry to detect GKLF at various stages of tumor progression in the breast, prostate, and colon. Overall, expression of GKLF mRNA was detected by in situ hybridization in 21 of 31 cases (68%) of carcinoma of the breast. Low-level expression of GKLF mRNA was observed in morphologically normal (uninvolved) breast epithelium adjacent to tumor cells. Increased expression was observed in neoplastic cells compared with adjacent uninvolved epithelium for 14 of 19 cases examined (74%). Ductal carcinoma in situ exhibited similar expression as invasive carcinoma, suggesting that GKLF is activated prior to invasion through the basement membrane. Expression as determined by Northern blot was increased in most breast tumor cell lines and in immortalized human mammary epithelial cells when these were compared with finite-life span human mammary epithelial cells. Alteration of GKLF expression was confirmed by the use of a novel monoclonal antibody that detected the protein in normal and neoplastic tissues in a distribution consistent with localization of the mRNA. In contrast to most breast tumors, expression of GKLF in tumor cells of colorectal or prostatic carcinomas was reduced or unaltered compared with normal epithelium. The results demonstrate that GKLF expression in epithelial compartments is altered in a tissue-type specific fashion during tumor progression, and suggest that increased expression of GKLF mRNA and protein may contribute to the malignant phenotype of breast tumors.

Membrane type I-matrix metalloproteinase-mediated degradation of type I collagen by oral squamous cell carcinoma cells.

Oral squamous cell carcinomas are highly invasive lesions that destroy adjacent tissues and invade bone and muscle, which is most likely the result of matrix metalloproteinase (MMP) activity. We examined three cell lines derived from squamous cell carcinoma of the tongue for their intrinsic capacities to degrade interstitial collagen with the goal of identifying the matrix-degrading enzymes. SCC-25 and SCC-15 cells degrade reconstituted fibrillar type I collagen in the absence of exogenous growth factors or cytokines when seeded as a colony on dried films. Degradation is confined to the subjacent matrix, is enhanced 2-3-fold by phorbol ester, and is strictly MMP-dependent, as it is blocked by BB-94 and tissue inhibitor of metalloproteinases-2 but not by inhibitors of serine and cysteine proteinases. Both cell lines express active (M(r) 57,000) membrane type I-MMP (MT1-MMP) on their surfaces, as detected by surface biotinylation and immunoprecipitation. Concomitantly, both cell lines activate endogenous MMP-2 when cultured on type I collagen films, as assessed by zymography. Phorbol ester treatment enhances collagen-induced MMP-2 activation, which is accompanied by the appearance of a surface-labeled M(r) 43,000 form of MT1-MMP. Treatment of cells with a synthetic furin inhibitor, which inhibits processing of the MT1-MMP zymogen, blocks collagen degradation. In contrast, CAL 27 cells do not degrade collagen under either basal or phorbol 12-myristate 13-acetate-stimulated conditions. Although proMT1-MMP (M(r) 63,000/65,000) is detectable in these cells by immunoblot analysis, they express greatly reduced levels of active MT1-MMP on their surfaces relative to SCC-25 and SCC-15 cells. Correspondingly, CAL 27 cells cultured on collagen express neither latent nor active gelatinases. Immunoblots of lysates and conditioned media revealed the constitutive expression of proMMP-1 and proMMP-13 in all three cell lines. We conclude that in the absence of exogenous growth factors or accessory stromal cells, degradation of interstitial collagen by oral squamous cell carcinoma cells requires a threshold level of active MT1-MMP on cell surfaces.

Training in metabolomics research. I. Designing the experiment, collecting and extracting samples and generating metabolomics data.

Metabolomics is perhaps the most challenging of the -omics fields, given the complexity of an organism's metabolome and the rapid rate at which it changes. When one sets out to study metabolism there are numerous dynamic variables that can influence metabolism that must be considered. Recognizing the experimental challenges confronting researchers who undertake metabolism studies, workshops like the one at University of Alabama at Birmingham have been established to offer instructional guidance. A summary of the UAB course training materials is being published as a two-part Special Feature Tutorial. In this month's Part I the authors discuss details of good experimental design and sample collection and handling. In an upcoming Part II, the authors discuss in detail the various aspects of data analysis.

Structural models of human apolipoprotein A-I: a critical analysis and review.

Human apolipoprotein (apo) A-I has been the subject of intense investigation because of its well-documented anti-atherogenic properties. About 70% of the protein found in high density lipoprotein complexes is apo A-I, a molecule that contains a series of highly homologous amphipathic alpha-helices. A number of significant experimental observations have allowed increasing sophisticated structural models for both the lipid-bound and the lipid-free forms of the apo A-I molecule to be tested critically. It seems clear, for example, that interactions between amphipathic domains in apo A-I may be crucial to understanding the dynamic nature of the molecule and the pathways by which the lipid-free molecule binds to lipid, both in a discoidal and a spherical particle. The state of the art of these structural studies is discussed and placed in context with current models and concepts of the physiological role of apo A-I and high-density lipoprotein in atherosclerosis and lipid metabolism.

Induction of collagenase-3 (MMP-13) in rheumatoid arthritis synovial fibroblasts.

There is a growing body of evidence that implicates matrix metalloproteinases (MMPs) as major players in numerous diseased conditions. The articular cartilage degradation that is characteristic of rheumatoid arthritis (RA) is believed to be mediated by the collagenase subfamily of matrix metalloproteinases. The preference of collagenase-3 (CL-3) for collagen type II makes it a likely candidate in the turnover of articular cartilage and a potential target for drug development. In this study, RA synovial membrane tissue was shown to express CL-3 mRNA by reverse transcriptase-polymerase chain reaction (RT-PCR) and protein by immunohistochemistry. Fibroblasts isolated and cultured from RA synovial membrane tissue were induced to express CL-3 mRNA. CL-3 mRNA was detected after PMA treatment in 16 of the 18 RA synovial membrane fibroblast cell lines established for this study. These fibroblasts also expressed mRNA for collagenase-1 (CL-1, MMP-1), membrane type-1 matrix metalloproteinase, gelatinase A, gelatinase B, stromelysin-1, stromelysin-2, TIMP-1, and TIMP-2. They were further shown to express CL-1 mRNA constitutively and CL-3 mRNA only after stimulation with PMA, IL-1, TGF-beta1, TNF-alpha, or IL-6 with IL-6sR. These fibroblasts also expressed after induction both CL-1 and CL-3 at the protein level as determined by Western blot analyses and immunofluorescence.

Glycosylation and NH2-terminal domain mutants of the tissue inhibitor of metalloproteinases-1 (TIMP-1).

Mutants in the tissue inhibitor of metalloproteinases-1 (TIMP-1) protein have been created by site-directed mutagenesis and expressed in HeLa cells, using a recombinant vaccinia virus system. Removal of either or both glycosylation sites yielded proteins which retained wild-type inhibitory activity against both human fibroblast-type collagenase (FIB-CL) and Mr 72000 gelatinase (GL). However, the double glycosylation mutant protein was expressed at a level that was 2-4-fold lower than that of the wild-type or the single site glycosylation mutants. The 'tiny-TIMP' COOH-terminal deletion mutant that lacks the last 57 residues was also inhibitory, but the dose-response curve suggested that the interaction with the Mr 72000 gelatinase had been altered. A number of replacement mutants in the highly conserved NH2-terminal domain, including replacement of P5A and P8A or a double mutation in the VIRAK sequence which is absolutely conserved in all TIMPs in all species (VIRAK to VIAAA), also yielded functional proteins capable of inhibiting FIB-CL and Mr 72000 GL and of forming SDS-resistant complexes with FIB-CL. None of the above manipulations abolished inhibitory function suggesting that binding of the inhibitor by the enzyme involves multiple interactions.

Trans complementation of an E1A-deleted adenovirus with codelivered E1A sequences to make recombinant adenoviral producer cells.

Replication-incompetent adenovirus is conventionally produced by cells that supply replication-enabling proteins from viral sequences present in trans. As an alternative means of recombinant adenovirus production, replication-enabling E1A sequences were cotransduced into human prostate carcinoma cells infected with an E1A-deleted adenovirus containing a luciferase expression cassette. The replication-enabling plasmid was cotransduced by ionic linkage to the recombinant adenovirus exterior. Cells cotransduced with the replication-enabling plasmid made new adenovirus with titers up to 8 x 10(6) in the supernatants 72-120 hr after transduction. Like the parent virus, the virus present in the cotransduced supernatants and lysates was capable of transferring luciferase activity to new cells. The virus present in the cotransduced cell supernatants was amplified and shown to be identical to the parent virus by genomic analysis. It was concluded that simultaneous addition of a replication-defective adenovirus and a replication-enabling gene sequence in a trans configuration converts some of the cotransduced cells into recombinant adenovirus-producing cells.

The nucleotide sequence of the polypeptide IX gene of human adenovirus type 3.

The nucleotide sequence of the DNA segment encompassing the polypeptide IX gene of class B human adenovirus serotype 3 (Ad3) has been determined using cloned restriction fragments. There is only a single, open translational reading frame capable of specifying a protein of 138 amino acids, comparable to the Mr 12 000-13 000 of protein IX detected in virions (Wadell, 1980). The corresponding region of a closely related class B virus, Ad7, is virtually identical (Dijkema et al., 1981), but the comparable segments of class C viruses Ad2 or Ad5 are much less homologous (Aleström et al., 1980; Maat et al., 1980). There are 150 single bp changes and 19 deletion-insertions, at least one frameshift, together affecting 210 nucleotides within the 455 bp comparison positions of the protein-coding regions of Ad2 (423 bp) and Ad3 (417 bp). Each of the 19 deletion-insertions involves an integral multiple of 3 bp in phase with the open translation frame. There is no "TATA" promoter box in Ad3 DNA at the position comparable to that of Ad2. The deduced protein sequences near the amino-terminus are extensively conserved between the two classes of viruses, but the carboxy-terminal portion and the nucleotide sequences flanking the gene are much more diverged. In both classes, these N- and C-terminal regions of the inferred proteins are linked by an alanine-rich chain, an arrangement suggestive of two functional domains.

The nucleotide sequence and protein-coding capability of the transposable element IS5.

The nucleotide sequence of IS5, a bacterial insertion sequence, has been determined. It is 1195 bp long and contains an inverted terminal repetition of 16 bp with one mismatch. One open reading frame, spanning nearly the entire length of the element, could encode a polypeptide of 338 amino acids. Upon insertion into a DNA segment, IS5 causes a duplication of 4 bp. Based on seven examples, this site of insertion appears to be nonrandom, and the consensus target site sequence is C . T/A . A . G/A (or C/T . T . A/T . G on the opposite strand). The nucleotide sequences of IS5 insertions into the B and cim genes of bacteriophage Mu have allowed tentative identification of the protein-coding frames of B and cim.

The nucleotide sequence of the gene encoding protein IVa2 in human adenovirus type 7.

The nucleotide sequences of cloned DNA segments encoding the IVa2 gene from Ad7 and a portion of Ad12 (group B and group A human adenoviruses, respectively) have been determined. When compared to Ad5, a group C adenovirus, these sequences have been found to be 80% homologous. Most changes are transitions or transversions. This high degree of nucleotide homology results in a high degree of amino acid conservation in the predicted polypeptides encoded from these genes; most nucleotide changes occur at the third position in the codon. The predicted polypeptide contains 448 amino acids and has a calculated Mr-value of 50700. The positions of the 5' end of the mRNA and of the donor and acceptor splice sites of Ad7 and Ad12 can be inferred by analogy to those of Ad5. A long open reading frame starting upstream from the IVa2 gene overlaps the N-terminal portion of the polypeptide but is encoded in a different reading frame. Within this overlapping region, the long open reading frame is more conserved in amino acid sequence than is the presumed IVa2 polypeptide, suggesting that evolutionary pressure was exerted on the longer protein, a product of viral early region 2B. The high degree of conservation of this E2B region within the overlapping segment suggests that its activities must be more important for adenovirus infection than are the functions encoded in the amino-terminus of the IVa2 gene.

Sequences of human adenovirus Ad3 and Ad7 DNAs encoding the promoter and first leader segment of late RNAs.

DNA segments containing the major promoter at coordinate 16.5 for rightward transcription from human adenovirus serotypes 3 and 7 (Ad3 and Ad7), two closely related class B viruses, have been sequenced and found virtually identical. Furthermore, over 80% of the nucleotides of Ad3 and Ad7 in this entire region are homologous to their counterparts in the DNA of the more distantly related class C serotype Ad2. There are the same number of nucleotide pairs among these serotypes within the region compared. Most changes are transitions or transversions and the several single-base deletions are always compensated by nearby insertions. These few changes nonetheless result in 24 differences between Ad7 (or Ad3) and Ad2 in a total of 32 cleavage sites. The promoter for the rightward-transcribed RNAs and the first segment of the consanguinous tripartite leader found at the 5'-ends of all the later mRNAs derived from that promoter have been identified by analogy to the nucleotide sequences of Ad2. In particular, the "Hogness box" or RNA polymerase staging site for the major rightward transcription unit is completely homologous to that of Ad2. There are only six bp changes in the first late leader segment despite previous evidence suggesting that they might be quite heterologous. A prominent dyad axis of symmetry exists just upstream from the presumed 5'-end of the late RNA. However, unlike the stem-loop structure proposed for Ad2 by Ziff and Evans (1978), the base changes relative to Ad2 mandate a different potential stemloop structure in the single strand of Ad3 and Ad7 DNAs. This hairpin places the "Hogness box" immediately next to the 5'-end of the RNA at the base of stem. An analogous dyad axis of symmetry or stemloop structure can be found in a number of eukaryotic systems, including the major rightward transcription unit of Ad2. This feature may be of relevance to the positioning of RNA polymerase II on the DNA and to the promotion of transcription.

The nucleotide sequence of the genes encoded in early region 2b of human adenovirus type 7.

The nucleotide sequence of a cloned DNA segment encoding the early region 2b from the group B human adenovirus Ad7 has been determined. When compared to Ad2, a group C adenovirus, these sequences were found to be approx. 80% homologous within the l-strand gene-coding regions. Most changes are transitions or transversions, although several deletions/insertions also occur within the N-terminal domain of one of the coding regions. The substantial nucleotide homology results in a high degree of amino acid conservation in the predicted polypeptides encoded by the early region 2b genes. Two major open reading frames, corresponding to the Mr 87000 and Mr 140000 polypeptides of Ad2, are found in the l strand of Ad7 between genome coordinates 28.5 to 23.1 and 13.8, respectively. The r strand of the DNA in this region encodes the three leader segments joined to the 5' end of the most late viral mRNAs, and also encodes the i-leader segment found between the second and third leaders on some mRNAs. The positions of the donor and acceptor splice sites of the three leaders are conserved and can be identified by homology to Ad2. Only two of the unidentified open reading frames (URF) in Ad2 (Gingeras et al., J. Biol. Chem., in press) can be found in Ad7. URF1, encoding an Mr 13500 polypeptide at genome coordinate 17, is predominantly conserved in nucleotide and amino acid sequence, but contains one half as many arginine amino acids as does URF1 of Ad2. URF2, encoding an Mr 13600 protein which lies within the i-leader region, is not well conserved in either nucleotide or amino acid sequence.

Restriction endonuclease BamHI cleaves bacteriophage Mu DNA within cistrons E and F.

We have cleaved phage Mu DNA with restriction endonucleases EcoRI and BamHI and have cloned three specific DNA fragments from the middle of the Mu genome into vector plasmid pBR322. By marker rescue experiments, we have determined that the two BamHI cleavage sites in Mu DNA occur within cistrons E and F.